This document summarizes research on the expression of E-cadherin and N-cadherin in cultures of bovine corneal epithelium and endothelium. It states that cadherins are important cell-cell adhesion proteins that form adherens junction complexes. The development of E-cadherin in corneal epithelial cells and N-cadherin in endothelial cells starts between 2-4 days after seeding the cells in culture, as shown through immunofluorescence and western blot analyses. Transepithelial electrical resistance measurements also increase significantly over the first 4-6 days as cadherins form adherens junctions between the cells.

1. Expression of E- and N- cadherin in cultures of bovine
corneal epithelium and endothelium

2.  A tissue-engineered cornea based on untransformed
corneal cells, should mimic the properties of the
native cornea.
 In addition to use as corneal replacement, a corneal
equivalent could be a useful model for studying
wound healing, physiology, toxicology, and
pharmacology.

3.  - Cadherins are a vast super family of cell-cell adhesion proteins.
 -calcium dependent cell membrane proteins and exist in cadherin-catenin
 complex. (adherens junction proteins)
 -with three subfamilies.
 -roles: cell-cell contact, Tissue morphogenesis, inhibits cancer development
http://www.hindawi.com/journals/vmi/2012/357187/fig1/

4.  -Corneal epithelium is a polarized tissue with distinct plasma membrane on its
apical and basolateral domains( dependent on adherens junction complexes).
 -N-cadherin is essential for maintaining proper structure of corneal endothelial
AJCs. Its ablation leads to increased endothelial permeability and corneal edema in
mature eyes.

5.  According to some experiments (Giasson et.al 2014) corneal equivalent by seeding
fibroblasts into a collagen gel on which epithelial and endothelial cells were added on
each side, cells from the epithelial and endothelial layers expressed adherens junction
proteins, indicating the presence of cell–cell contacts.
Mol Vis. 2014; 20: 386–394.
Published online 2014 Mar 29.

6.  The development of, E-cadherin and N-cadherin in
corneal epithelial and endothelial cell culture ,starts in
2-4 days after seeding of the corneal epithelial and
endothelial cells .

7.  1-Separation and isolation of bovine corneal cells
 2-subculture and feeding the cells
 3- Passage of epithelium and endothelial cells
 4-transfer the cells to 6-well
 5-they were lifted to the liquid-air interface
 6-TER measurement
7-Treatment of membrane and immunofluorescence
 8-SDS-PAGE and western blots*


10.  Measurement of electrical resistance
 Electrical resistance measurements as a measurement of cell-
cell junctions, was used to determine the situation of
adherence junctions of the cells.

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 Int Poster J Dent Oral Med 3 (2001), No.
2 (15.06.2001)

11.  IF and WB and TER will be repeated at 2, 4,6 and days
after seeding
 Each experiment will be repeated 3 or 4 times
 Observation of IF and WB at these times: cadherins
formed?
 TER: Mean Resistance at each time will be plotted.
 Differences in mean resistance will be tested for
statistical significance with a repeated measure one
way analysis of variance (time)(ANOVA).

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13.  Cadherins and a-,b-catenins will be increasingly
expressed with time after seeding of epi and endo cells
by observation of IF and Western blots
 N- and E-cadherins will be only expressed in
endothelial and epithelial corneal cells, respectively
 Cadherins and catenins are expressed after 3-4 days
after seeding of corneal epithelial and endothelial cells
in monoculture

14.  1. Nelson WJ, Adams CL, Chen YT, Smith SJ. Epithelial cell polarity from the outside looking in. News
Physiol Sci. 2003;18:143–6. [PMC free article] [PubMed]
 2-Vassil S. Vassilev, Michiko Mandai, Shigenobu Yonemura, Masatoshi Takeichi .Loss of N-Cadherin from
the Endothelium Causes Stromal Edema and Epithelial Dysgenesis in the Mouse Cornea.Investigative
Ophthalmology & Visual Science October 2012, Vol.53, 7183-7193. doi:10.1167/iovs.12-9949
 3-Claud J.Giasson, Alexandre Deschambeault, Patrick Carrier and Luice Germain. Adherens junction
proteins are expressed in collagen corneal equivalents produced in vitro with human cells. Mol Vis. 2014;
20: 386–394.
 Published online 2014 Mar 29.